Chimeric cDNAs which contained portions of both wild-type and attenuated hepatitis A virus (HAV) cDNA were constructed. Plus strand RNAs transcribed from constructs were transfected into primate cells to identify those regions of the genome critical for in vitro cultivation. Chimeric viruses harvested from productive transfection experiments were then inoculated into marmosets in order to assess virulence. Infectivity assays of the chimeric genomes demonstrated that genes within the P2 region determine the ability of the virus to grow efficiently in vitro. Site-specific mutagenesis indicates that two of the genes in this region are both involved. Virulence assays suggest that attenuation for animals and growth in tissue culture are closely coupled. In order to determine if the phenotypes of virulence and in vitro cultivation co-vary or can be dissociated, attempts will be made to grow virulent virus from the wild-type clone by using engineered complementing cell lines or by determining the absolute minimum number of mutations required for growth in vitro.